1. Field of the Invention
The present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to the prolongation of gene therapy and the inhibition of inflammation decreasing the activity of TNF.
2. Description of the Related Art
The immune response to adenovirus vectors has been recognized as a limiting factor in the successful application of gene therapies. Cellular and humoral immune responses have been implicated in the shortened time-span of transgene expression, the eradication of transfected cells, and the preclusion of the opportunity to re-administer the adenovirus (Yang et al.,1996b; Kass-Eisler et al.,1996; Yang et al., 1996c; Yang and Wilson, 1995c). Immunosuppressive drugs, including cyclophosphamide, FK506, and cyclosporine reduce this immune response (Jooss et al.,1996; Vilquin et al., 1995). Other strategies reported to control the immune response include reduction of the T-cell response by anti-T cell therapy (Sawchuk et al.,1996) and anti-CD4 monoclonal antibodies (Dematteo et al.,1996), reduction of co-stimulatory activity using CTLA4-Ig (Guerette et al., 1996; He et al., 1996; Kay et al., 1997), and induction of intrathymic tolerance (Ilan et al., 1996). B-cell activation production of neutralizing antibody production is decreased after treatment with anti-CD40 (Yang et al., 1996a) and deoxyyspergualin (Smith et al., 1996). In addition, modification of adenovirus vectors may be effective in reducing the immune response (Gao et al., 1996; Fisher et al., 1997).
Antigen presenting cells (APCs) play an important role in both specific and non-specific immune responses to adenovirus. Processing of the antigen, presentation of the antigen in the context of major histocompatibility complex (MHC) class I molecules, and cytokine production by the antigen presenting cells contribute to the immune response to adenovirus (Yang et al., 1995a; Schowalter et al., 1997). The E19 adenovirus gene product leads to decreased MHC class I antigen expression and an immune response to this gene product occurs in class I mutant mice indicating that MHC class I antigen expression is not required.
Interleukin-12 (IL-12) and other cytokines, such as interferon-xcex3 (IFN-xcex3), are expressed at high levels early after adenovirus infection and it has been proposed that these evoke a TH1-type response to adenovirus (Yang et al., 1995b; Coutelier et al., 1995; Day et al., 1994). The role of TNF-xcex1 production by antigen presenting cells in the early inflammatory immune response to adenovirus is less well understood (Smith et al., 1994). TNF-xcex1 increases expression of the adenovirus E3 proteins, which, in turn, protects the cells from the lytic activity of TNF-xcex1. These finding demonstrate the central role for neutralization of TNF-xcex1 for the virus to survive (Deryckere et al., 1995; Tufariello et al., 1994; Ranheim et al., 1993; Korner et al., 1992; Wold, 1993).
TNF-xcex1 acts by crosslinking either the high affinity receptor p55 TNF-RI or the low affinity p75 TNF-RII, the TNF-RI being most closely associated with inflammatory responses (Smith et al., 1994). The effectiveness of TNF-xcex1 antagonists, such as sTNF receptor (sTNFR) inhibitors, in ameliorating inflammation of the lung has been demonstrated in acute lung injury, which frequently develops following hemorrhage and is characterized by increased proinflammatory cytokine levels and massive neutrophil accumulation in the lung (Abraham et al., 1994; Su et al., 1998; Rabinovici et al., 1996). A rapid increase in the level of TNF-xcex1 mRNA expression among pulmonary cell populations precedes the development of acute lung injury. In mice, therapy with sTNFR:Fc, composed of soluble dimeric human p80 TNF-R linked to the Fc region of human IgG1, prevents the post-hemorrhage increases in circulating and pulmonary TNF-xcex1 levels and diminishes the increase in IL1xcex2, IL6, TNF-xcex1 and IFN-xcex3 mRNA (Haak-Frendscho et al., 1994; Fisher et al., 1996; Wooley et al.,1993; Kolls et al.,1994).
A novel TNF-binding protein (TNF-bp) has been developed which is composed of a polyethylene glycol (PEG)-linked dimer of the recombinant human TNF-RI soluble receptor, and is capable of binding to TNF-xcex1 with high affinity (Evans et al.,1996). The use of the TNF-bp PEG-linked dimer has potential therapeutic advantages over the use of monomeric TNF-bp. First, dimerization of TNF-bp enhances the avidity for TNF-xcex1, and the resultant mobilization of two sites within the TNF-xcex1 trimer may prevent TNF-xcex1-mediated signal transduction since cross-linking of two TNF-Rs by TNF is required to trigger cellular responses (Evans et al., 1996). Second, the PEG-linked dimer has a large molecular mass with a predictably slow clearance, which allows relatively infrequent dosing. Third, the PEG-linked dimer may exhibit lower immunogenicity than the pure protein since it has been reported that PEG conjugation to proteins leads to lowered antibody response (Delgado et al., 1992; Katre 1990).
The prior art is deficient in the lack of effective means for the prolongation of gene therapy expression. The present invention fulfills this longstanding need and desire in the art.
The present studies indicate that there is greatly decreased inflammatory disease and prolonged gene therapy expression in AdCMVlacZ virus-infected mice treated with TNF-bp compared with vehicle-treated mice. These results indicate that TNF-xcex1 is a key factor in the pathogenesis of inflammation in AdCMVlacZ-virus infected mice. Thus, TNF-bp PEG-linked dimer may be therapeutically useful in reducing the inflammatory response to adenovirus gene therapy.
The clinical application of adenoviral gene therapy is impeded currently by the potent host immune response to the virus that limits the duration of its effects. These studies showed the role of TNF-xcex1 on the inflammatory response and expression of a lacZ expressing adenovirus in the lung and liver of mice after administration of recombinant adenovirus using soluble TNF receptor (TNF-bp). The mononuclear cell inflammatory response was determined histologically at different times after intranasal or intravenous administration of a recombinant adenovirus expressing the lacZmarker gene. The expression of the activity of the lacZ gene product of the adenovirus. Treatment with TNF-bp reduced circulating levels of TNF-xcex1, greatly reduced the inflammatory response, and resulted in prolonged expression of lacZ for up to 30 days in the lung and liver after either intranasal or intravenous administration of adenovirus. Both intranasal and intravenous administration of adenovirus to vehicle-treated C57BL/6-+/+ mice resulted in a persistent inflammatory response and clearance of the adenovirus in the lung. However, clearance of the virus also was observed in the liver, despite a minimal inflammatory response. These results indicate that TNF-xcex1 is the primary factor driving the inflammatory response and leading to elimination of adenovirus-infected cells in the lung and liver as TNF-bp was capable of inhibiting these effects.
In one embodiment of the present invention, there is provided a method of increasing adenoviral gene expression in a tissue of an animal, comprising the step of administering to said animal a pharmacologically effective dose of tumor necrosis factor binding protein.
In another embodiment of the present invention, there is provided a method of reducing an inflammatory response associated with adenoviral administration in a tissue of an animal, comprising the step of administering to said animal a pharmacologically effective dose of tumor necrosis factor binding protein.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.